Mutagenicity of N,N'-dimethylurea and methylamine hydrochloride in the Ames Salmonella/microsome test: absence of mutagenic response.

Methyl isocyanate (MIC) in aqueous solution forms methylamine (MA) and N,N'-dimethylurea (DMU). MA in buffered system further converts into its salt form, methylamine hydrochloride (MAH). Therefore, MAH and DMU were evaluated for their mutagenic activity in the in vitro Ames Salmonella/microsome mutagenicity test. The liquid preincubation protocol was followed, using tester strains TA98, TA100 and TA104 of Salmonella typhimurium, in the presence of 0, 5, 15 and 30% Aroclor 1254-induced rat liver S9 mixture. DMU and MAH did not induce a mutagenic response in any of the tester strains, both in the presence and in the absence of S9 mixture. The results therefore confirm that MIC in its native form or as its unknown metabolites is responsible for the mutagenic activity reported earlier by us in the his tester strains TA100 and TA104 of Salmonella typhimurium (Mutation Res., 204 (1988) 123-129) and not due to its hydrolysis products, MA or DMU.     

Evaluation of the mutagenic potential of endosulfan using the Salmonella/mammalian microsome assay

The mutagenic potential of endosulfan, a polychlorinated insecticide, was assessed using the highly sensitive Salmonella tester strains TA97(a), TA98, TA100 and TA102. It exhibited a toxic effect at dose levels of 50 micrograms/plate and higher. Plate incorporation studies did not show mutagenic response with any of the tester strains used. A modification of the assay using a preincubation procedure showed mutagenic activity with and without metabolic activation with TA97(a) only. Increased toxicity was observed after addition of phenobarbital-induced S9 mix.     

Structure-function characterization for ethidium photoaffinity labels as mutagens in salmonella

The development of photoaffinity probes to characterize the binding process and subsequent biological activity of a drug has recently been emphasized by the synthesis of two ethidium azide analogs. The initial findings showed that one of the azido analogs, the 8-azido-3-amino derivative, was at least 40-fold more mutagenic and toxic in Salmonella tester strain TA1538 than the other analog, the 3,8-diazido derivative. These observations suggested the need to examine the structural requirements of ethidium photoaffinity labels for frameshift mutagenic activity in Salmonella. Thus, the isomer of the monoazide, the 3-azido-8-amino derivative, and two deaminated monoazide derivatives were synthesized and all of the ethidium analogs were screened in two Salmonella frameshift tester strains, TA1537 and TA1538, and in their excision-repair positive isogenic strains. The results presented in this paper demonstrate that two substituents are needed to produce significant mutagenicity and toxicity by the compound. One substituent, usually the amino group, is required for mutagenic activity, perhaps by orienting the phenanthridinium ring into its mutagenic configuration. The other substituent, the azido group, is required for covalent attachment, a requisite for mutagenic activity.Thus, photoaffinity labeling has provided a means of comparing structure with mutagenic activity for ethidium compounds.     

Lack of Mutagenicity of the Fungicide 2,4,5,6-Tetrachloroisophthalonitrile in the Ames Salmonella/Microsome Test

The fungicide 2,4,5,6-tetrachloroisophthalonitrile was found to be not mutagenic to five Salmonella typhimurium tester strains. Incorporation of either a liver or a kidney activation system did not increase the mutagenic activity of the fungicide.     

Mutagenicity of aminophenyl and nitrophenyl ethers, sulfides, and disulfides.

The mutagenic activity of several aromatic amines and aromatic nitro compounds related to 4,4'-methylenedianiline towards Salmonella typhymurium tester strains TA100 and TA98 was evaluated. The heteroatomic analogs of 4,4'-methylenedianiline which include aminophenyl and nitrophenyl ethers, sulfides and disulfides were assayed in the presence of rat-liver homogenate. The relative mutagenic response of these analogs indicated the following order of activity, --S-- greater than --O-- greater than --CH2--CH2-- greater than or equal to --S--S--. In both tester strains 4-aminophenylsulfone was inactive with and without microsomal activation. The p-nitrophenyl ethers, sulfides and disulfides were relatively strong mutagens without microsomal activation towards TA100. While 4-nitrophenyldisulfide was found to possess significantly different mutagenic activity than 4-nitrothiophenol in TA98, 4-AMINOPHENYl disulfide has similar mutagenic properties to 4-aminothiophenol in both tester straains TA100 and TA98.     

Changes in mutagenicity of protein pyrolyzates by reaction with nitrite.

Pyrolyzates of protein and related materials were treated with nitrite under acidic conditions, and the mutagenic activity toward Salmonella tester strains was determined. After treatment with nitrite in acidic solution, casein pyrolyzate, an extract of roasted chicken meat, tobacco-smoke condensate and some aromatic amines showed appreciable decreases in their mutagenic activities toward Salmonella typhimurium TA 98. Aromatic amines in the pyrolyzates may be changed by nitrite treatment to other forms having no or lower mutagenic activity toward Salmonella typhimurium TA 98. The contribution by aromatic amines to the total mutagenic activity of the pyrolyzates was as high as 80% in both casein pyrolyzate and extract of roasted chicken meat and 50% in tobacco-smoke condensate. Pyrolyzates of protein and related materials did not show a decrease in the mutagenic activity toward Salmonella typhimurium TA 100 with the same treatment.     

Mutagenicity of N-acetyl and N,N'-diacetyl derivatives of 3 aromatic amines used as epoxy-resin hardeners

3 epoxy-resin hardeners, 4,4'-diaminodiphenyl ether (DDE), 4,4'-diaminodiphenylmethane (DDM), and 4,4'-diaminodiphenylsulfone (DDS), and their N-acetyl and N,N'-diacetyl derivatives were examined for their mutagenicity using Salmonella typhimurium TA98 and TA100 as the tester stains and an S9 mix containing a rat-liver 9000 X g supernatant fraction as the metabolic activation system. DDE and DDM were mutagenic towards TA98 and TA100 in the presence of S9 mix while DDS exhibited no significant mutagenic activity towards these tester strains. These epoxy-resin hardeners were metabolized in vivo and their N-acetyl and N,N'-diacetyl metabolites were found in the urine. Among these acetyl metabolites, only N-acetyl-DDE was found to be mutagenic towards TA98 and TA100 in the presence of S9 mix. None of these acetyl metabolites exhibited significant mutagenic activity towards these tester strains in the absence of S9 mix.     

Mutagenicity and DNA-damaging activity for several pesticides tested with Bacillus subtilis mutants

The active pure compounds of 4 pesticides were tested for DNA-damaging and mutagenic activity in Bacillus subtilis and Salmonella typhimurium tester strains. Included were zinc ethylenebisdithiocarbamate (dithane), 1,2-dihydropyridazine-3,6-dione (maleic hydrazide), O,O-dimethylphosphorodithioate (malathion), and 1,2-dibromoethane (fumazone). These agents gave either weak or negative mutagenic responses with the Salmonella/microsome tests for mutagenicity, but were all positive when the tester was B. subtilis strain TKJ6321. Of the 4 chemicals, only fumazone required metabolic activation with rat-liver S9 mix. Upon activation, it produced a volatile mutagenic product. Dithane, maleic hydrazide, and malathion were all mutagenic and did not require metabolic activation. Among these agents, dithane was strongly mutagenic while fumazone, maleic hydrazide and malathion were moderately mutagenic. Only dithane gave significant DNA-damaging activity when applied to a battery of repair-deficient B. subtilis mutants. For the chemicals reported, it is concluded that B. subtilis is superior to S. typhimurium in the detection of mutagenic activity. We strongly recommend its use for prescreening procedures in combination with the S. typhimurium testers.     

Aliphatic halogenated hydrocarbons produce volatile Salmonella mutagens

Production of volatile mutagenic metabolites from 5 halogenated promutagens was examined by a simple modification of the conventional Salmonella/microsome mutagenicity assay. This method incorporates the taping together of 2 agar plates face to face during the initial portion of their incubation at 37 degrees C. By varying the contents of the soft agar in each of the two plates with respect to promutagen, S9 and tester strain cells, mutagenesis due to volatile promutagens and their metabolites could be quantitated separately. Using the taped plate assay, volatile mutagenic metabolites were detected from the promutagens 3-(2-chloroethoxy)-1,2-dichloropropene, the herbicides diallate, triallate and sulfallate, and the flame-retardant tris-(2,3-dibromopropyl) phosphate (Tris-BP). All compounds except Tris-BP were also found to be volatile promutagens. The mutagenic metabolites accounted for 50-80% of the activity of these compounds observed in the standard assay. Morever, our studies suggest that a small, but appreciable percentage of the mutagenic metabolites from all 5 compounds escaped detection in the conventional, untaped assay. Mutagenic activity of the volatile mutagenic metabolites from diallate was quenched by various Salmonella tester strains independent of their responsiveness to diallate mutagenesis. Detection of volatile mutagen formation from diallate was also prevented by cysteine and glutathione, but not by DNA or metyrapone. This taped plate method for the Salmonella assay should facilitate future investigations of the detection, isolation and identification of volatile mutagenic metabolites from other promutagenic compounds or mixtures.     

Mutagenic activity of nine N,N-disubstituted hydrazines in the Salmonella/mammalian microsome assay.

The mutagenic activity of N,N-dimethyl-, N,N-diethyl-, N,N-dibutyl-, N,N-diisobutyl-, N,N-di(p-tolyl)-, N-ethyl-N-phenyl-, N,N-dibenzyl-, N,N-diphenyl- and N,N-diisopropylhydrazine was examined in the Salmonella/mammalian microsome assay using the strains TA1535, TA1537, TA97, TA98, TA100, TA102 and TA1530. All nine hydrazines were mutagenic in at least one tester strain, although of borderline significance for some of the compounds. The mutagenic potencies of the hydrazines varied 2-3 orders of magnitude, from very weak to moderate mutagenic activity. In general, the addition of S9 resulted in a lowering of the mutagenic activity and a lowering of the toxic properties of the hydrazines. The test results were relatively difficult to evaluate due to toxic effects of many of the test compounds on the test bacteria which may have resulted in an underestimation of the mutagenic potencies of some of the compounds. The pattern of mutagenic activity of the hydrazines in the different tester strains indicates that more than one mechanism of action may be involved in the mutagenicity.     

Mutagenic evaluation of etintidine (BL-5641), a novel histamine H2-receptor antagonist, using reverse mutation tests in bacteria and forward mutation tests in V79 Chinese hamster cells

The mutagenic effects of etintidine (BL-5641), a novel histamine H2-receptor antagonist, were assessed using mutation in Salmonella typhimurium, Escherichia coli, and V79 Chinese hamster cells (V79 cells). Etintidine did not increase the revertant colonies in the presence or absence of S9 mix at concentrations from 5 to 5000 micrograms/plate in either of the bacterial tester strains. Etintidine also did not increase 6-thioguanine- and ouabain-resistant colonies in V79 cells in the presence or absence of S9 mix even at 37% cellular survival concentration. There was no evidence that etintidine had any mutagenic activity in any of the tests.     

Mutagenicity of the coccidiostat diaveridine in the Salmonella/mammalian microsome assay

The coccidiostat diaveridine was tested for mutagenicity in the Salmonella/microsome assay with tester strains TA100 and TA98. This compound was not mutagenic in either tester strain in the presence and absence of rat S9 mix, but was found to be mutagenic in strain TA100 after metabolic activation with hamster S9 mix.     

Mutagenicity of 4-aminoantipyrine and 4-nitrosoantipyrine, the C-nitroso derivative of antipyrine

The drug antipyrine and its 4-substituted analogs, 4-aminoantipyrine, 4-dimethylaminoantipyrine (aminopyrine) and 4-nitrosoantipyrine were tested for mutagenicity against the screening array of Salmonella typhimurium tester strains TA100, TA98, TA97, TA102 and TA104. Antipyrine and aminopyrine were nonmutagenic to all 5 tester strains even in the presence of S9. 4-Aminoantipyrine was directly mutagenic to TA97 only and the presence of S9 slightly increased its activity. 4-Nitrosoantipyrine was directly mutagenic to all tester strains used and S9 decreased its activity except with strain TA102. The possible long-term hazards of C-nitroso compounds derived from drugs and dietary constituents are discussed in view of their pluripotent direct genotoxicity.     

A new Salmonella tester strain,TA97, for the detection of frameshift mutagens: A run of cytosines as a mutational hot-spot

We have developed a new Salmonella tester strain, TA97, for use in the Salmonella/microsome mutagenicity test. DNA sequencing has shown that this strain contains and added cytosine, resulting in a run of six cytosines at the mutated site in the histidine D gene. Its mutagenic specificity is similar to that of the frameshift mutagen tester strain, TA1537, which also contains an added cytosine in a run of cytosines and is currently among the five standard tester strains used for general mutagen screening. We assessed the mutagenic potency of 21 frameshift mutagens for TA1537 and TA97. TA97 was considerably more sensitive than TA1537 to reversion by these frameshift mutagens. In addition, one agent, PR toxin (from Penicillium roqueforti), which was not detected by any of the previously existing standard tester strains, did revert TA97; and two substituted aryl-alkyl triazenes, which had not been reported previously to be frameshift mutagens, were mutagenic in this new tester strain. We suggest that TA1537 be replaced by TA97 for general screening of mutagenicity.     

Mutagenicity of polycyclic aromatic compounds (PAC) identified in source emissions and ambient air

Several polycyclic aromatic compounds (PAC) including nitrated and oxygenated derivatives of polycyclic aromatic hydrocarbons (PAH) were tested for mutagenic activity in the Salmonella/microsome assay. Among the compounds tested the isomer mix of nitro-1-hydroxypyrenes showed the highest direct mutagenic response in both the Salmonella strain TA98 and TA100 (1251 revertants/micrograms and 463 revertants/micrograms, respectively). The direct-acting mutagenicity of the nitro-1-hydroxypyrene isomer mix was dependent upon reduction of the nitro function as evidenced by the decrease in activity observed with the nitroreductase-deficient and arylhydroxylamine esterifying-deficient tester strains. The oxygenated derivatives of PAH containing aldehyde or keto groups showed weak or no mutagenic responses. In most cases addition of S9 was essential for any mutagenic activity and the strain TA100 was more sensitive than the strain TA98. Within this group, 7H-dibenzo[c,g]fluoren-7-one showed the highest mutagenic effect; 7 and 22 revertants/micrograms using the strains TA98 and TA100, respectively.     

Salmonella/mammalian microsome assay with tetranitromethane and 3-nitro-L-tyrosine

The nitrosating agent tetranitromethane (TNM) and the nitrosation product 3-nitro-L-tyrosine (NT) were tested for mutagenic activity in the Salmonella/mammalian microsome assay. TNM showed strong genotoxic activity: it was mutagenic in all tester strains used (TA97, TA98, TA100, and TA102). The maximum mutagenic activity was reached between 16 and 32 micrograms/plate using the standard plate test; higher amounts led to distinct bactericidal effects. The mutagenicity was independent of an in vitro activation system. In the preincubation assay an increased bactericidal effect was observed. In contrast to TNM, NT, the nitrosation product, was non-mutagenic and non-toxic in the standard plate test and with the preincubation method up to 5000 micrograms/plate with and without S9 mix and with all tester strains used. Although TNM is a strong direct-acting mutagen, its nitrosating effect on proteins does lead to nongenotoxic nitro products of tyrosine in proteins.     

Mutagenicity of methyl isocyanate in the modified test conditions of Ames Salmonella/microsome liquid-preincubation procedure

Methyl isocyanate (MIC) was tested for mutagenicity using the Ames Salmonella/microsome liquid-preincubation procedure with slight modification of test conditions. In the modification the preincubation mixture was incubated at 10 degrees C for 60 min. MIC was assayed both in the presence and absence of Aroclor-1254-induced S9, using 5 tester strains of Salmonella typhimurium, TA97a, TA98, TA100, TA102 and TA104. MIC induced mutagenic response in two base-pair substitution strains, TA100 and TA104, in the presence and absence of S9. However, mutagenic response in the presence of S9 was low as compared to that in the absence of S9. In the comparative mutagenic activity at 3 different preincubation test conditions (37 degrees C for 20 min, 20 degrees C for 40 min and 10 degrees C for 60 min), optimum mutagenic response was observed at 10 degrees C for the 60-min test condition. However, no mutagenic response was observed at 37 degrees C for the 20-min test condition.     

Activation of promutagenic chemicals by Streptomyces griseus containing cytochrome P-450soy

Streptomyces griseus cells containing cytochrome P-450soy oxidize a diverse array of xenobiotic compounds. This metabolic capability was exploited for activation of promutagenic chemicals such as polycyclic aromatic hydrocarbons, aromatic amines and small aliphatics in a modified Salmonella/Ames plate incorporation assay using tester strains TA98 and TA1538. In this assay promutagens such as 3,3'-dimethylbenzidine, 3,3'-dimethoxybenzidine, benzidine, 2-acetylaminofluorene, 2-aminoanthracene, 2,4-diaminotoluene, 4-aminobiphenyl, benzo(a)pyrene, chloropicrin and N-nitrosodimethylamine were oxidized to mutagenic metabolites by S. griseus intact cells which mutated Salmonella tester strains (TA98 and TA1538). S. griseus failed to activate 7,12-dimethylbenzanthracene and 4-chloro-2-nitroaniline. In parallel tests performed with rat liver homogenate (S9), N-nitrosodimethylamine was not activated.     

Dietary factors affecting the urinary mutagenicity assay system. II. The absence of mutagenic activity in human urine following consumption of red wine or grape juice

The mutagenic activity of urine samples from nonsmoking individuals before and after the consumption of either red wine or grape juice was determined. Urine samples collected from individuals on liquid or regular diets were concentrated using XAD-2 resin. No mutagenic activity of urine concentrates was detected with Salmonella tester strains TA98 or TA100 with or without microsomal activation. The addition of 1000 units of beta-glucuronidase into the agar overlay did not show any mutagenic activity. The mutagens in red wine and grape juice, however, were extracted using the XAD-2 column. Concentrates of urine samples spiked with either of the two extracts exhibited mutagenic activity.     

Mutagenic activity of certain synthetic steroids: structural requirement for the mutagenic activity in Salmonella and E. coli

Eight steroids, structurally related to cholesterol, were tested for mutagenic activity in the Ames tester strains. All the test compounds were mutagenic without metabolic activation, although metabolic activation markedly enhanced their activity. A significant decrease in the survival of the radiation-sensitive mutants recA and lexA of Escherichia coli was observed as compared to their wild-type counterpart in the presence of the steroids. The role of recA and lexA genes gains further support from the lambda prophage induction in the lysogen as well as with Salmonella strains triggering the error-prone SOS response. Structural features which appear to be essential for mutagenic activity in these strains of the steroids are (1) reactive thio, sulfonyl or sulfinyl groups at the 6 position and (2) a halogen group at the 3 position of the steroidal nucleus. The mutagenicity appears to involve the formation of H2O2 as well as superoxide and hydroxyl radicals.